Purification of His-tagged hepatitis B core antigen from unclarified bacterial homogenate using immobilized metal affinity-expanded bed adsorption chromatography

Wei Boon Yap, Beng Ti Tey, Noorjahan Banu Mohamed Alitheen, Wen Siang Tan

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Hepatitis B core antigen (HBcAg) is used as a diagnostic reagent for the detection of hepatitis B virus infection. In this study, immobilized metal affinity-expanded bed adsorption chromatography (IMA-EBAC) was employed to purify N-terminally His-tagged HBcAg from unclarified bacterial homogenate. Streamline Chelating was used as the adsorbent and the batch adsorption experiment showed that the optimal binding pH of His-tagged HBcAg was 8.0 with a binding capacity of 1.8mg per ml of adsorbent. The optimal elution condition for the elution of His-tagged HBcAg from the adsorbent was at pH 7 in the presence of 500mM imidazole and 1.5. M NaCl. The IMA-EBAC has successfully recovered 56% of His-tagged HBcAg from the unclarified E. coli homogenate with a purification factor of 3.64. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of the recovered His-tagged HBcAg was not affected throughout the IMA-EBAC purification process and electron microscopy revealed that the protein assembled into virus-like particles (VLP).

Original languageEnglish
Pages (from-to)3473-3480
Number of pages8
JournalJournal of Chromatography A
Volume1217
Issue number21
DOIs
Publication statusPublished - May 2010
Externally publishedYes

Fingerprint

Hepatitis B Core Antigens
Chromatography
Adsorption
Purification
Metals
Adsorbents
Viruses
Immunosorbents
Virus Diseases
Chelation
Hepatitis B virus
Virion
Escherichia coli
Electron microscopy
Assays
Electron Microscopy
Enzyme-Linked Immunosorbent Assay
Enzymes
Proteins

Keywords

  • His-tagged HBcAg
  • IMA-EBAC
  • Streamline Chelating
  • Unclarified bacterial homogenate
  • Virus-like particles

ASJC Scopus subject areas

  • Analytical Chemistry
  • Organic Chemistry
  • Biochemistry

Cite this

Purification of His-tagged hepatitis B core antigen from unclarified bacterial homogenate using immobilized metal affinity-expanded bed adsorption chromatography. / Yap, Wei Boon; Tey, Beng Ti; Alitheen, Noorjahan Banu Mohamed; Tan, Wen Siang.

In: Journal of Chromatography A, Vol. 1217, No. 21, 05.2010, p. 3473-3480.

Research output: Contribution to journalArticle

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